Plant hormones act in a complex network where their pathways regulate and interact to control different mechanisms, such as development and stress responses. This crosstalk between hormones can be exploited by pathogens to suppress plant defense responses and thereby increase pathogen growth. Pseudomonas syringae pathogenicity is reliant on a Type III secretion system (TTSS) that acts as a specialized injection apparatus to deliver virulence proteins, known as type III effectors (TTEs), into the plant cell cytosol. In my work, I have screened hormone inducible promoter::GUS transgenic Arabidopsis thaliana lines against a P. syringae TTE library in order to identify TTEs involved in the perturbation of hormone signaling in planta. Through this screen I identified two P. syringae TTEs, HopAK1 and HopAL1, both belonging to the same bacterial strain P. syringae pv. maculicola ES4326. I found that HopAK1 can sensitize A. thaliana plants to auxin. On the other hand, HopAL1 activates auxin signaling. Monitoring of auxin signaling was done using transgenic DR5::GUS plants. Both TTEs render the plant susceptible to bacterial infection, highlighting a potential relationship between increased auxin signaling and virulence.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-20T13:00:17Z No. of bitstreams: 1 texto completo.pdf: 327861 bytes, checksum: 10b3c4ac9c1fb002cc94c4da4da28d0e (MD5) / Made available in DSpace on 2017-04-20T13:00:17Z (GMT). No. of bitstreams: 1 texto completo.pdf: 327861 bytes, checksum: 10b3c4ac9c1fb002cc94c4da4da28d0e (MD5) Previous issue date: 2002-12-03 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A busca de alimentos produzidos sob um sistema de manejo menos agressivo ao meio ambiente vem sendo adotado por um número cada vez maior de produtores. Entretanto, apesar de existirem diversos benefícios na redução ou até eliminação do uso de defensivos, a grande diversidade de doenças em tomateiro (Lycopersicon esculentum Mill.), capazes de limitar a produção, torna necessária a busca por alternativas viáveis, eficientes e tecnicamente comprovadas. Dentre os organismos mais estudados, bactérias têm sido relatadas como agentes de biocontrole capazes de atuar por meio de mecanismos como antibiose, parasitismo, competição e indução de resistência. O presente trabalho teve como objetivos selecionar bactérias do filoplano do tomateiro, baseado em uma estratégia de seleção in vivo, verificando se há um método de isolamento que permita obter antagonistas eficientes no controle da pinta-preta, causada por Alternaria solani, requeima por Phytophthora infestans, mancha-bacteriana pequena por Pseudomonas syringae pv. tomato e mancha-bacteriana por Xanthomonas vesicatoria. Objetivou ainda estudar a possibilidade dos mecanismos de antibiose e indução de resistência serem responsáveis pelo controle destas doenças e se testes de antibiose in vitro são adequados como critério de seleção. Caracterizar aspectos biológicos dos antagonistas que podem otimizar sua aplicação como agente de biocontrole. Determinar a quais produtos antimicrobianos os isolados são insensíveis, visando fornecer subsídios para o desenvolvimento de meios semi-seletivos e estudos de dinâmica populacional. Verificar sua compatibilidade com antibióticos e fungicidas registrados para o controle de enfermidades do tomateiro, a fim de inseri-lo no sistema de manejo integrado e estudar a eficiência de antagonistas selecionados em condições de campo. Os resultados demonstram que, em folíolos mais jovens, os métodos de isolamento que visam obter bactérias da população total e da superfície do filoplano, foram os que permitiram obter a maioria dos antagonistas. O único obtido de folíolos mais velhos foi proveniente da população capaz de habitar sítios protegidos do filoplano e/ou resistir a fatores de estresse. Não se observou relação entre características biológicas dos antagonistas e dos patógenos testados. Nos testes de antibiose com os antagonistas selecionados, o isolado UFV-STB 6 foi capaz de produzir compostos voláteis e inibir a germinação de cistos de Phytophthora infestans, o que possivelmente deve estar envolvido no controle da requeima. O isolado UFV-IEA 6 produziu quitinase, havendo uma tendência em reduzir a taxa de crescimento de Alternaria solani por compostos voláteis. Ficou demonstrado que os testes de antibiose in vitro são inadequados como critério para seleção de agentes de biocontrole do filoplano de tomateiro. A caracterização dos melhores antagonistas demonstrou que três são bactérias Gram-positivas, em forma de bastonete, e uma Gram-negativa, pleiomórfica. Dentre as Gram-positivas todas são anaeróbias facultativas e uma forma endósporos. Nenhum antagonista foi capaz de causar reação de hipersensibilidade (HR) em fumo e produzir pigmento fluorescente in vitro. Os períodos de geração calculados a partir das curvas de crescimento revelaram que três isolados são capazes de se multiplicar rapidamente em meio de cultura, o que é uma característica desejável. Os resultados obtidos a partir dos antibiogramas, mostraram que existem antibióticos que podem ser utilizados para elaboração de meios semi-seletivos, adequados a cada antagonista e os testes de compatibilidade com antibióticos e fungicidas utilizados na cultura do tomateiro revelaram que os antagonistas podem ser expostos aos fungicidas benomyl, enxofre, dimetomorph e tiofanato-metílico. Verificou-se também a inadequação de se utilizarem compostos antimicrobianos em meio de cultura para isolamento de agentes bacterianos de controle biológico, uma vez que os antagonistas selecionados foram sensíveis à maioria dos produtos testados. Os testes com as enzimas indicadoras do estado de indução de resistência, β-1,3-glucanases, Fenilalanina amônia-liase (PAL), Peroxidases (PO), Polifenoloxidases (PPO) e Lipoxigenases (LOX), indicaram que o isolado UFV-IEA 6 foi capaz de promover aumento significativo na atividade das PO, evidenciando a possibilidade do antagonista agir como indutor de resistência. Esse parece ser o primeiro caso que se tem conhecimento de uma bactéria não fitopatogênica do filoplano induzindo resistência na mesma cultura de onde foi obtida. Os testes com os dois antagonistas em condições de campo demonstraram que UFV-STB 6 foi o mais eficiente em reduzir a severidade da requeima no terços médio e superior das plantas, enquanto UFV-IEA 6, somente no terço superior. Houve tendência na redução do progresso da septoriose por UFV-STB 6 e capacidade em diminuir o número de frutos com sintomas de requeima. Os resultados demonstram o potencial de uso dos agentes de biocontrole selecionados para as doenças da parte aérea de tomateiro estudadas. / Farmers are increasingly adapting environmentally less aggressive management systems for food production and there are many benefits in reducing or even eliminating pesticide use. Due to a large number of production limiting diseases on tomato (Lycopersicon esculentum), it is desirable to find technically viable and proven alternatives for their control. Among the microorganisms, bacteria have been reported as biocontrol agents capable of acting through antibiosis, parasitism, competition and induced resistance. The present study aimed at selecting the tomato phylloplane bacteria, based on in vivo isolation strategy, and to determine if this method permits obtaining efficient antagonists to control Alternaria solani leaf spot, Phytophthora infestans blight, small bacterial leaf spot caused by Pseudomonas syringe pv tomato, and bacterial leaf spot caused by Xanthomonas vesicatoria. The study also aimed at determining mechanisms of action, such as antibiosis and induced resistance, involved in disease control, and to determine if the antibiosis tests are sufficient selection criteria. The study also included biological characterization of the antagonists that may optimize their use. To help develop selective or semi-selective media for population dynamic studies, insensitivity of selected isolates to some antimicrobial compounds was also determined. The compatibility of select antagonists with antibiotics and fungicides registered for control of tomato diseases was elucidated so that the antagonist can be inserted in the integrated management. Field studies were done to determine the efficiency of select antagonists. The isolation method that obtain total bacterial population from the phylloplane of the young leaflets permitted obtaining maximum number of antagonists. The only one isolate obtained from the older leaflets originated from the population capable of inhabiting protected sites of phylloplane and/or that resist stress factors. There was no relation between biological characteristics of the antagonists and of the pathogen tested. In the antibiosis testes, the isolate UFV-STB 6 produced volatile compounds that inhibited germination of P. infestance cyst and may be involved in the control of blight. The isolate UFV-IEA 6 produced chitinase and showed a tendency to reduce A. solani growth by the volatile compounds. In vitro antibiosis testes were inadequate criteria to select biocontrol agents from tomato phylloplane. The characterization of promising antagonists showed that three were Gram positive bacilli and one was gram negative pleiomorphic bacteria. Among the Gram positives all were facultative anaerobes and the one formed endospores. None of the antagonists caused hypersensitive reaction (HR) in tobacco, and did not produce fluorescent pigment in vitro. The generation period calculated from the growth curve revealed that three isolates are capable of multiplying rapidly in the culture media, which is a desirable characteristic. The antibiograms showed that there are antibiotics that can be used for elaboration of semi-selective media for each of antagonists and the compatibility testes with antibiotics and fungicides used on tomato crop revealed that the antagonists can be exposed to fungicides such as benomyl, sulfur, dimethomorph and thiophante-methyl. Many antimicrobial compounds were inhibitory to the selecte antagonists in culture media used for isolation of bacterial biocontrol agents, therefore were inadequate for use in selective media. The analysis of enzymes involved in induced resistance, like -1,3-gluconase, phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and lipoxigenase (LOX), showed that the isolate UFV-IEA 6 was capable of increasing PO activity, showing the possibility of being a resistance inducer . This appears to be the first case of a non-pathogenic phylloplane bacterium inducing resistance in a plant of origin. The field testes with two antagonists, UFV-STB 6 was more efficient in reducing the blight severity in the middle and upper third of the plant, while UFV-IEA 6 only in the upper third. The latter isolate also showed a tendency for reducing the Septoria leaf spot progress and the number of fruits with the blight symptoms. The results showed these isolates have the potential of use to control tomato diseases of aerial parts. / Tese importada do Alexandria

Doctor of Philosophy / Department of Plant Pathology / Xiaoyan Tang / P. syringae is a group of bacterial phytopathogens that can infect a wide variety of plants. These bacteria rely on the type III secretion system (TTSS) to deliver effectors into plant cells for infection. The TTSS genes, that encode the TTSS apparatus and the effectors, are repressed when bacteria grow in nutrient rich media but are strongly induced in the plants and in minimal medium (MM). Plant cutin monomers appear to negatively regulate the P. syringae TTSS genes. It is poorly understood how bacteria sense the environmental signals to regulate the TTSS genes. By genetic screen, four sets of transposon insertion mutants displaying aberrant TTSS gene expression were isolated: KB and fin mutants derepress the TTSS genes in rich medium KB and in the presence of a cutin monomer precursor in MM, respectively; min and pin mutants are defective in induction of TTSS genes in MM and in plants, respectively. A putative two-component sensor histidine kinase, RohS, is identified to be required for the induction of avrPto-LUC in MM and in plants. The rohS gene is in an operon containing a two-component response regulator gene rohR. Mutation of rohS in P. s. phaseolicola and P. s. tomato reduced the bacterial pathogenicity on hosts and HR-inducing activity on non-hosts. Our results suggested that RohS acts upstream of HrpR/HrpS. The phosphorylated RohR represses TTSS genes. It is likely that RohS acts as phosphatase of RohR in the TTSS-inducing conditions, and subsequently derepresses TTSS genes. Simple sugars such as glucose, sucrose and fructose are known to be inducers of the TTSS genes. Isolation of four min mutants defective in fructose-uptake enabled us to study if sugars serve as extracellular signals or as essential nutrients. Our results suggest that fructose acts as an essential nutrient for the activation of type III genes. These mutants slightly compromised induction of avrPto promoter in Arabidopsis and pathogenicity on the host bean plant, but displayed normal HR elicitation on non-host plant tobacco. The reduced pathogenicity suggested that exploitation of fructose from the host tissue is an important means for pathogenesis of P. s. phaseolicola.

Doctor of Philosophy / Department of Plant Pathology / Jianmin Zhou / Plants activate innate immune responses or innate immunity upon pathogen infection. There are two types of plant innate immunity: PAMP-triggered innate immunity (PTI) and effector-triggered innate immunity (ETI). The molecular basis for ETI has been well documented. However, the study on PTI and its interplay with pathogen virulence is in its infancy. My research focuses on the interplay between PTI and bacterial virulence in Pseudomonas-Arabidopsis interactions. NHO1, a gene required for nonhost resistance to Pseudomonas syringae, encodes for the 3-glycerol kinase in Arabidopsis genome. NHO1 functions, at least in part, by depriving glycerol from nonhost bacteria cells. NHO1 is induced by a well-known bacteria PAMP flg22. The induction of NHO1 correlates well with the resistance against Pseudomonas syringae pv. tabaci because a mutant strain of P. s. pv. tabaci deficient in NHO1 induction gains partial virulence on Arabidopsis plants. P. s. pv. tomato strain DC3000 induces transient NHO1 expression that is suppressed in a type III secrection system-dependent manner. Using protoplast assay, nine DC3000 effectors that are able to suppress NHO1 were identified. One of them, HopAI1, induces leaf chlorosis and helps nonpathogenic bacterial growth when expressed in Arabidopsis plants, suggesting that HopAI1 has virulence activity in planta. To study AvrB virulence activity in Arabidopsis plants, one mutant compromised in AvrB-specific RAR2.6 induction has been characterized in detail. rrb3 is more susceptible to a nonhost bacteria P. s. pv. tabaci strain 6505, a virulent bacteria P. s. pv. tomato strain DC3000 and an avirulent bacteria strain DC3000 (avrB). The mutant allele rrb3 carries a point mutation at the end of RAR1 CHORD II domain. RRB3 (RAR1), together with NDR1, is involved in the type II nonhost resistance to P. s. pv. tabaci but not in the type I nonhost resistance to P. s. pv. phaseolicola. RAR1 participates in basal resistance against DC3000 by antagonizing COI1 activity. AvrB targets RAR1 to trigger AvrB-dependent leaf chlorosis and enhanced bacterial growth. The AvrB-dependent enhanced bacterial growth but not leaf chlorosis requires COI1, suggesting that AvrB targets JA signaling pathway to promote parasitism.

Les plantes dans leur milieu naturel, sont constamment soumises à de multiples contraintes environnementales de nature biotique ou abiotique qui sont à l'origine de nombreuses pertes de rendement. Afin de répondre à ces stress, les plantes mettent en place des réponses adaptées qui reposent sur l'activation de voies de signalisations. L'une des voies importantes est celle impliquant la phosphorylation des protéines par les Mitogen Activated Protein Kinase (MAPKs). La régulation de l'activité des MAPKs est indispensable et dépend en partie de protéines phosphatases telles que les MAPK Phosphatases (MKPs). Ce travail a consisté à étudier les mécanismes de régulation de l'activité phosphatase de TMKP1, la seule MKP connue de blé dur, par les calmodulines et les protéines 14-3-3. Dans un premier temps, nous avons montré que l'activité phosphatase de TMKP1 est stimulée en présence des ions K+, Li+, Mg 2+ et surtout par le Mn2+. Des expériences de GST pull dawn ont permis de mettre en évidence une interaction, calcium dépendante, entre TMKP1 et la calmoduline (CaM) et nous avons démontré que l'activité de TMKP1 est inhibée par le complexe CaM/Ca2+. En revanche, ce même complexe stimule l'activité de TMKP1 en présence des ions Mn 2+. Ce mode de régulation d'une MKP par CaM/Ca2+ dépendant des ions Mo2+ est décrit ici pour la première fois. Dans un second temps, la présence au niveau de la séquence de TMKP1 d'un domaine de liaison aux protéines 14-3-3s, nous a incité à mener des essais d'immunoprécipitation à l'issue desquelles nous avons montré que TMKP1 pourrait interagir avec les 14-3-3s chez le Blé et que celles-ci stimulent l'activité phosphatase de TMKP1 in vitro. Enfin, l'implication de TMKP1 dans la réponse des plantes aux stress biotiques et abiotiques a été évaluée. Les tests de gennination ont révélé que la sur-expression de TMKP1 chez Arabidopsis permet une meilleure tolérance à la salinité. Ces mêmes plantes semblent également être plus résistantes à une infection bactérienne causée par Pseudomonas syringae que des plantes sauvages. Ces données suggèrent que TMKP1 agirait comme régulateur positif de la réponse d'Arabidopsis au stress salin et à l'infection P. syringae. L'ensemble des résultats obtenus ici a permis de dévoiler de nouvelles propriétés jamais décrites chez une MKP végétale et offre une nouvelle vision sur le rôle de ces phosphatases dans le contrôle des voies de signalisations MAPKs impliquées dans la réponse des plantes aux stress de l'environnement. / Due to their sessile lifestyle, plants are constantly subjected to a variety of biotic and abiotic stresses causing tremendous yield losses. To survive under these conditions, plants have evolved different sigualing pathways allowing efficient stress responses. The Mitogen Activated Protein Kinase (MAPKs) are key sigual transduction molecules, which respond to varions external stimuli. However, the activity of MAPKs has to be strictly regulated by protein phosphatase such as MAPK Phosphatase (MKPs). ln the present study we describe the regulation ofTMKPI, a wheat MKP, by Calmodulins (CaM) and 14-3-3 proteins. We fust showed that phosphatase activity oTMKP1 is stimulated by K\ Li\ Mg2+ and especially by Mo2+. Using GST pull dawn assays, we demonstrated that TMKP1 binds to CaM in a Ca2+-dependent manner. Moreover, the CaM/Ca2+ complex inhibits the catalytic activity of TMKP1 in a CaM-dose dependent marmer. However, in the presence of Mo2+, this activity is enhanced by CaM/Ca2+ complex. Such effects were not reported so far, and raise a possible role for CaM and Mo 2+ in the regulation of plant MKPs during cellular response to extemal siguals. Moreover, a fine sequence analysis ofTMKPl revealed the presence of a conserved 14-3-3 mode 1 binding site. This finding incited us to perfonn co-immunoprecipitation assays on wheat protein extracts through which we provide proof-of-concept evidence for interaction of TMKP1 with 14-3-3 proteins. Interestingly, the phosphatase activity of TMKP1 was shawn to be enhanced by several plant and yeast 14-3-3 isoforms. Finally, the involvement of TMKP1 in plant stress responses was evaluated. Our data showed that the overexpression of TMKP1 in Arabidopsis resulted in a higher tolerance to salt stresses and to bacterial infection caused by P. syringae. Such findings suggest thal TMKP1 may act as a positive regulator of Arabidopsis responses to salt stresses and P. syringae infection. All together, our results provide novel functional properties for plant MKPs, and should add new knowledge to our understanding of the raie of these phosphatases in the control of MAPK signaling pathways controlling plant responses to various environmental stresses.

The aim of this diploma thesis is determination of the influence of deficient nutrition in combination of Pseudomonas syringae pv. tomato infection on tomato (Solanum lycopersicum L.) growth and development. In hydroponic cultivation experiment, tomato variety Darinka was cultivated in solutions without N, P, K, Ca, Mg and in variant with all nutrition (control variant). The dry matter of root and stem was weighed and root length, density and electrical capacitance were set. The content of nutrition in plants was also assessed. The level of infection was determined in 1st term, the highest level had plants with insufficient of Ca and P. High reduction of root and stem dry matter was found in Ca, N, P and Mg deficient plants. The low root electrical capacitance, length and density was determined in plants with Ca, N a P deficiency within infected group and in Ca and N deficient plants within noninfected group. Bacterial infection caused the reduction of all observing parameters in contrast to the plants from noninfected group. The antagonism and synergism between the elements were also showed.

Plant innate immunity is activated upon pathogen attack by recognizing their avirulent (avr) genes by Resistant (R) genes leading to R-gene resistance or host resistance. Another form of innate immunity is non-host resistance that is exhibited by a given plant species to most strains of a microbial species. R-gene resistance activates salicylic acid (SA) that is synthesized from methyl salicylic acid (MeSA) by Salicylic Acid Binding Protein 2 (SABP2). It was hypothesized that SABP2 plays the similar role in non-host resistance also. Growth experiments and non-host related gene analysis experiments were conducted on tobacco plants using P.s tabaci and P.s. phaseolicola that are host and non-host pathogens on tobacco respectively. Tobacco control plant C3 that expresses SABP2 and 1-2 that is RNAi silenced in SABP2 expression were used in this study. Results suggest that SABP2 may not have any significant role in tobacco non-host resistance.

No description available.

<p>Systemic Acquired Resistance (SAR) is a plant defense response induced by an initial infection in one part of the plant that leads to broad-spectrum resistance to normally virulent pathogens in distant naïve leaves. As part of the Cameron research team, I contributed to demonstrating that the lipid transfer protein, DIR1 is required for SAR long distance signaling in <em>Arabidopsis</em> and travels from induced to distant tissues during SAR. A highly similar<em> Arabidopsis</em> protein DIR1-like was identified and is thought to be responsible for the occasional SAR-competent phenotype observed in the <em>dir1-1</em> mutant. This work provides evidence for the idea that DIR1 and DIR1-like are paralogs created by a recent duplication event and that similar to DIR1, DIR1-like may travel to distant tissues during SAR. To better understand DIR1 and DIR1-like contribution during SAR, <em>dir1-1dir1-like</em> double mutant transgenic plants were created as well as transgenic plants expressing epitope- (HA and FLAG) and fluorescent- (iLOV and phiLOV) tagged DIR1 and DIR1-like to facilitate visualization of movement during SAR. Several putative DIR1 orthologs were identified in crop plants and cucumber CucDIR1 was shown to be functionally equivalent to AtDIR1 in <em>dir1-1</em> complementation studies providing further evidence that DIR1 plays an important role in SAR across plant species. By analyzing conservation between DIR1, DIR1-like and the putative DIR1 orthologs, several protein residues were identified that may be important for DIR1 function during SAR. DIR1 proteins were modified at these sites and the importance of these residues was supported by the reduced binding of the TNS hydrophobic probe in these DIR1 variants. Taken together, this thesis suggests that DIR1 and DIR1-like both participate in SAR in <em>Arabidopsis</em>, that DIR1 crop orthologs are also important for the SAR response and that DIR1 possesses several sites that are critical for its function in long distance SAR signaling.</p> / Doctor of Philosophy (PhD)

<p><em>Eutrema salsugineum</em> is an extremophile plant native to the Yukon Territory and coastal China. As an extremophile, Yukon <em>Eutrema</em> is tolerant to highly saline, drought conditions and cold temperatures while Shandong <em>Eutrema</em> can survive in highly saline conditions (Griffith et al., 2007; Guevara et al., 2012; Inan et al., 2004). The disease resistance responses of the Yukon and Shandong accessions of <em>Eutrema</em> were investigated to understand how an abiotic stress-tolerant plant responds to biotic stress. A pathosystem was developed using <em>Pseudomonas</em> <em>syringae</em> pv. <em>tomato</em> DC3000 (<em>Pst</em>) to examine <em>Eutrema</em> defense responses. Compared to <em>Arabidopsis </em>(Col-0), both <em>Eutrema</em> accessions exhibited resistance to <em>Pst,</em> with Shandong <em>Eutrema</em> displaying greater resistance than Yukon <em>Eutrema</em>. Resistance to <em>P. syringae</em> pv. <em>maculicola</em> (<em>Psm</em>) was also observed in both accessions. Furthermore, both <em>Eutrema</em> accessions displayed a differential capacity for effector-triggered immunity (ETI). RNA-Seq data of uninoculated Shandong vs. Yukon <em>Eutrema</em> revealed an overrepresentation of defense genes including <em>PR1</em> (<em>pathogenesis-related1</em>; Champigny et al., 2013). Expression of the <em>Eutrema</em> <em>PR1</em> ortholog in uninoculated Shandong leaves combined with enhanced resistance to <em>Pst</em> compared to Yukon <em>Eutrema</em> or Col-0 <em>Arabidopsis</em> suggests that Shandong plants exist in a defense-primed state. Resistance to other pathogens such as <em>Pectobacterium</em> <em>carotovorum</em> ssp. <em>wasabiae</em> (<em>Pcw</em>) further supported the hypothesis that Shandong <em>Eutrema</em> is primed for pathogen tolerance. The <em>Eutrema</em>-<em>P. syringae</em> pathosystem will facilitate future studies to understand how <em>Eutrema</em> deals with multiple or concurrent stresses and this knowledge will contribute to efforts to improve tolerance to both abiotic and biotic stress in crop plants.</p> / Master of Science (MSc)